Enrichment of Stem Cells from Adult Tissues

ABSTRACT

Subjecting a heterogeneous cell population (one with both stem cells and non-stem cells) to extreme stress selectively eliminated the non-stem cells and resulted in the enrichment of stem cells in the population. The stress can take many forms, including without limitation, cell toxins, high temperature, high salt, and low oxygen (hypoxic) conditions. The number of stem cells remaining after stress were increased, and showed increased expression of traditional stem cell markers. The stem cells were shown to be capable of proliferation and differentiation into multiple types of cells. This method allows purification of stem cells from adult heterogeneous cell populations on a large scale basis without requirement of expensive equipment, and without requiring the presence of cell surface markers. Stem cells produced by the above method can be used for clinical applications, including tissue engineering.

This is a continuation of co-pending patent application Ser. No.12/842,754, filed Jul. 23, 2010, now allowed with the issue fee paid;which claimed the benefit of U.S. provisional patent application Ser.No. 61/228,245, filed Jul. 24, 2009.

This invention was made with government support under grant no.DE008911-16 and DE008911-17, awarded by the National Institutes ofHealth. The government has certain rights in this invention.

This invention pertains to methods and kits to enrich the number ofadult stem cells in a heterogeneous cell population and to isolate adultstem cells by subjecting the heterogeneous cell population to one ormore extreme stresses, such as high temperature, high osmotic fluids,and known cell toxins.

Stem Cells. Use of stem cells to regenerate or repair damaged ordefective tissues is a promising strategy to cure many human diseasesand disorders. Stem cells can be classified into embryonic stem cells,cord-blood-derived embryonic like stem cells and adult stem cells basedon their sources. Embryonic stem cells are from early embryos and arepluripotent. Adult stem cells are undifferentiated cells foundthroughout the body. Current culture techniques suggest that adult stemcells are multipotent; i.e., they can only differentiate to limited celltypes under current techniques although some adult stem cells have shownplasticity (see review by Raff 2003). A major advantage of using adultstem cells is that the patient's own cells could be isolated, expandedand induced to differentiate or regenerate for that same patient. Inturn, the cells derived or tissue regenerated from the stem cells wouldnot be rejected by the patient's immune system upon transplantation.

One major challenge to overcome in the application of adult stem celltherapy is to be able to identify, isolate and purify the adult stemcells and then proliferate sufficient quantities of the stem cells.Purification of stem cells can be very difficult because of theirrarity. For example, in primary isolated dental pulp cells, 0.2%-0.7%cells are colony-forming cells, which are putative stem cells (Gronthoset al., 2000). Therefore, for application of stem cell technology it isnecessary to develop efficient and reliable techniques that can isolateprimary stem cells from adult tissues. Because adult stem cells may onlydivide limited times in vitro; e.g; 80% of stem cells from human dentalpulp could not proliferate over 20 population doublings (Gronthos etal., 2002), starting with a pure population of such cells is necessaryfor obtaining an adequate number for subsequent differentiation andtissue engineering.

Stem cells were originally thought to be present only in organs thathave high cell turnover rates such as blood, skin, gut, and testis, butit is likely that most, if not all, adult organs contain stem cells. Inthat vein, stem cells have been identified or isolated from variousdental tissues including dental pulp (DPSCs), periodontal ligament anddental follicle (Gronthos et al., 2000; Seo et al., 2004; Morsczeck etal., 2005; Iohara et al., 2006; Jo et al., 2007). When cultured toothbud cells from 4 day postnatal rats were implanted onto scaffolds, toothtissues were generated (Duailibi et al., 2004). Dental pulp appears tocontain subpopulations of stem cells because single cell coloniesderived from pulp cells show diversity in cell surface markers (Gronthoset al., 2000). DPSCs have capabilities to form odontoblast-like andosteoblast-like cells (Braut et al., 2003; Mina and Braut 2004). Thus,stem cells exists in dental tissues with the possibility of using themin stem cell therapy and tissue engineering of dental tissues in vivo(review by Shi et al., 2005).

The dental follicle (DF), a loose connective tissue sac that surroundsthe unerupted tooth, plays different roles in the life of a tooth. Itspresence is required for eruption (Cahill and Marks, 1980; Marks andCahill, 1984) whereby it appears to regulate the osteoclastogenesis andosteogenesis needed for eruption (e.g., see review by Wise and King,2008; Wise et al., 2005; Wise and Yao, 2006). As the tooth pierces thegingiva, the DF differentiates into the periodontal ligament (PDL) toanchor the tooth in its socket to the surrounding alveolar bone. Inaddition to this differentiation of the DF to form the fibroblasts ofthe PDL, some DF cells are thought to differentiate into thecementoblasts of the tooth, as well as perhaps some of the osteoblastsof the alveolar bone (e.g., see review by Bosshardt and Schroeder,1996). Although some studies suggest that some of the cementoblasts mayarise from Hertwig's epithelial root sheath, as well as from the DF(Zeichner-David et al., 2003), other studies indicate that all of thecementoblasts arise from the DF (Diekwisch, 2001).

Stem cells appear to be present in the DF of human wisdom teeth(Morsczeck et al., 2005) and in the mouse DF (Luan et al., 2006). ThreeDF cell lines were cloned, one of which likely was the fibroblastic DFcells that form the PDL, another that remained undifferentiated, and athird that exhibited mineralization behavior as seen by von Kossastaining Injection of bovine dental follicle cells into immunodeficientmice results in the formation of cementum, as detected by ananti-cementum attachment protein (Handa et al., 2002). More recently, acementoblast progenitor cell line from bovine DF cells has been isolatedand immortalized (Saito et al., 2005).

According to the International Society for Stem Cell Research, dentalpulp stem cells (DPSCs) are believed to be “younger” than other adultstem cells, which means that they can be maintained in culture for alonger period of time, and they can differentiate into a variety of celltypes such as neuronal and adipose tissue (Gronthos et al., 2002; reviewby Liu 2006). DPSCs presented more striking odontogenic capability thanbone marrow stromal stem cells (Yu et al., 2007). Another advantage isthat the dental pulp is an accessible tissue source in deciduous teethshed by children. Stem cells have also been isolated from humanexfoliated deciduous teeth, i.e., SHED (Miura et al., 2004), and thesestem cells were able to repair calvarial defects (Seo et al., 2007). Thedental pulp can be collected at that time and patient-specific stemcells can be purified from the pulp, expanded and stored for future usesfor the given individual. In addition, the pulp can also be obtainedfrom extracted teeth.

Isolation of Stem Cells. Isolation of stem cells from dental tissues hasoften relied on their adherent and colony-forming properties on aculture surface. For example, such colony-forming method has been usedto isolate pulp stem cells from human third molars (Gronthos et al.,2000), and from human exfoliated deciduous teeth (Miura et al., 2004).Such isolation techniques are labor-intensive, time-consuming andskill-demanding because the cloning rings have to be placed over thecolonies one by one under a microscope. In addition, the colonyformation requires 2 weeks incubation time (Gronthos et al., 2000;review by Liu et al., 2006). In addition, the isolation did not yield auniform stem cell population; i.e., colonies derived from heterogeneousdental pulp cells show diversity in cell surface markers (Gronthos etal., 2000).

To select a pure population of dental pulp stem cells, immunomagneticbead sorting has been employed. Briefly, the technique involved bindingof an antibody of a stem cell marker to the potential stem cells, thenbinding of IgG-conjugated magnetic beads to the primary antibody,followed by collecting of the bead-positive cells (review by Liu et al.,2006). For application of the technique, stem cell specific surfacemarkers must exist. If no unique stem cell-specific surface marker ispresent, selection must be repeated with multiple markers, which greatlyincreases the cost while reducing the efficacy of the method.

Other techniques used to purify stem cells may be based on cell size(Hung et al., 2002); adherent properties to tissue culture surface(review by Owen & Friedenstein 1988; Caplan 1991; Kuznetsov et al.,1997; Pittenger et al., 1999); or marker dependent sorting such asfluorescence-activated cell sorting (FACS). Since both stem cells andnon-stem cells vary in sizes and adherent ability, isolation of stemcells based on size and adherent properties are not specific enough toobtain a pure population of stem cells (Vacanti et al., 2001; Young etal., 2004; Leong et al., 2004). For example, adherent single cellderived colonies of progenitors isolated from bone marrow display a widevariation in cell morphology and growth potential (review by Bianco andRobey 2000). In marker dependent sorting, the problem often encounteredis the lack of specific and easily detectable markers. Furthermore,reliable FACS sometimes requires large-frame ion lasers. For example,for sorting of side population stem cells, an expensive UV laser isrequired. In addition, the high fluid pressure during sorting mightdamage the biological activity of the sensitive cells, as discussed byGangopadhyay et al., (2004). Thus, development of a simple, inexpensiveand precise technique of isolating a pure population of stem cells isneeded.

In an injury or diseased tissue, the existing stem cells can act as arepairing mechanism. Stem cells possess some unique properties. Forexample, ABC transporters are one of the largest and most ancientprotein pumps responsible for the efflux of many molecules such as theDNA binding dyes, rhodamine and Hoechst from cells (Goodell et al.,1996; Wolf et al., 1993; Zhou et al., 2001). Effluxes of those dyesprevent or reduce the toxicity of the given chemical to the cells.Members of the ABC transporter super-family, BCRP1 and MDR1(P-glycoprotein), have been identified as stem cell markers because asignificantly higher level of these proteins are sited on the membraneof stem cells. Studies also indicate that stem cells produce high levelsof aldehyde dehydrogenases (ALDHs) to detoxify aldehydes (Cai et al.,2004; Storms et al., 1999).

Substrates of BCRP and ALDHs have been used to develop flow cytometrybased methods to isolate stem cells from various tissues. When Hoechst33342 is added to a heterogeneous population of cells, cells with ahigher level of BCRP, such as stem cells, will efflux more molecules ofthe dye, and thus result in a dull staining of their nuclei (Goodell etal., 1996). Such cells can be sorted out as a population, termed “sidepopulation,” by flow cytometry. Side population cells have been isolatedfrom multiple tissues including hematopoietic compartments (Goodell etal., 1997; Uchida et al., 2001), skeletal muscle (Gussoni et al., 1999;Jackson et al., 1999), brain (Hulspas et al., 2000) and dental pulp(Iohara et al., 2006). Additionally, embryonic stem cells also show sidepopulation phenotype (Zhou et al., 2001), indicating they are alsoequipped with a high level of BCRP pumps. Side population cells havebeen proved to be highly enriched for stem cells or primitive stem cells(Lin and Goodell 2006; Wolf et al., 1993; Leemhuis et al., 1996; Redvers2006, Parmar et al., 2003).

Similarly, stem cells have been identified based on the ability toefflux other fluorescence dyes that are substrates, such as rhodamine123, of ABC transporters (Orlic et al., 1993). However, as statedearlier, isolation of side population cells requires expensive flowcytometry with a special UV. Another limitation is that only thesubstrates that have distinct fluorescence emission can be used.Substrates that are highly efficient for the enzymes, and thus of lowtoxicity to the stem cells, can not be used if they are non-fluorescent.This limitation also applies to other types of substrates, such as thesubstrates of ALDHs. Some of the known substrates of ABC transportersare listed in Table 1 (review by Doyle and Ross 2003).

TABLE 1 Some substrates or drugs of ABC transporters MitoxantroneIdarubicinol topoisomerase I Pheophorbide a Protoporphyrin TopotecanFlavopiridol Zidovudine Daunorubicin Prazocin lrinotecan, SN-38 CIIO33lamivudine Epirubicin lndolocarbazole 9-Aminocamptothecin BBR3390Rhodamine 123 Hoechst 33342 Prazocin- Doxorubicin (DOX) MethotrexateLysotracker

ALDHs are a group of intracellular enzymes that catalyze oxidationreactions to convert cytotoxic aldehyde to non-toxic or low-toxiccarboxylic acid, thus resulting in cellular detoxification. ALDHs playan important role in the metabolism of ethanol, vitamin A, andcyclophosphamide. Recent advances in ALDHs research suggested that stemcells share the common characteristic of having a high level of ALDHs(Cai et al., 2004; Storms et al., 1999). For example, hematopoieticprogenitors, hematopoietic stem cells (HSC) and intestinal crypt stemcells display high levels of cytosolic ALDHs. Accordingly, those cellsare relatively resistant to cyclophosphamide (Kastan et al., 1990; Russoet al., 1989). This high ALDHs activity also appears to relate to thecolony-forming ability of stem cells over time (Lioznov et al., 2005).

ALDHs were thought to be telltale markers that distinguish stem cellsfrom other non-stem cells. The fluorescent substrates of the enzymes,such as AldeFluor, have been used in flow cytometry-based isolation topurify stem cells from multiple tissues, including neural stem cellsfrom brain (Corti et al., 2006) and hematopoietic stem cells from cordblood (Hess et al., 2005). ALDHs have a broad substrate list includingacetaldehyde and many biogenic amines.

Humans and other mammals maintain a constant body temperature at 37° C.in most tissues and organs. Alteration of body temperature, especiallyfever, is a frequently occurring symptom in many medical conditions.Fever may have evolved as a host defense mechanism through millions ofyears of evolution (review by Schaffner 2006; Soszynski 2003). Elevatedbody temperature creates a hostile environment for some pathogens(Fischler and Reinhart, 1997). However, elevated temperature changes themicro-environment in the cytoplasm, and affects many biochemicalprocesses. In extreme circumstances, it can impair cell functions andcause cell death (Harris, 1967). Some cells produce heat shock proteinsto prevent cellular damage by heat stress (Li et al., 1992). A heatshock protein, HSP105, has been shown to protect cancer cells fromapoptosis induced by heat shock (Hosaka et al., 2006). Evidence suggeststhat cancer cells are derived from the transformation or mutation ofnormal stem cells (review by Reya et al., 2001).

Another stress of cells often encountered is hypoxia in which severe andprolonged hypoxia can result in cell injury and death (Lennon et al.,2001). A study of cell survival in hypoxic conditions reported thatBCRP−/− (lack of functional BCRP) progenitor cells are more susceptibleto hypoxic conditions than are their counterpart wild type BCRP+/+ cells(Krishnamurthy et al., 2004). The protective effect of BCRP is theresult of its specifically binding to heme to prevent porphyrinsaccumulation. Such accumulation is detrimental to cells under hypoxicconditions. Additionally, hypoxia enhances BCRP expression to fortifysuch effect. When isolated mesenchymal stem cells were subjected inhypoxic conditions (5% O2), they proliferated more rapidly and formedmore colonies than they did in normal (20% O2) conditions (Lennon etal., 2001).

We have discovered that subjecting a heterogeneous cell population (onewith both stem cells and non-stem cells) to extreme stress selectivelyeliminates the non-stem cells and thus results in the enrichment of stemcells in the population. The stress can take many forms, includingwithout limitation, cell toxins, high temperature, high salt, and lowoxygen (hypoxic) conditions. We have shown that a greater percentage ofthe cells remaining after stress are stem cells based on traditionalstem cell markers. We have also shown that these cells are capable ofproliferation and differentiation into multiple types of cells. Thismethod allows purification of stem cells from adult heterogeneous cellpopulations on a large scale basis without requirement of expensiveequipment, and without requiring the presence of cell surface markers.Stem cells produced by the above method can be used for clinicalapplications, including tissue engineering.

We have identified stem cells in the dental follicles (DF) of the ratfirst mandibular molar by Hoechst staining, alkaline phosphatasestaining, and expression of side population stem cell markers. Thesecells were shown to be able to differentiate into osteoblasts,adipocytes and neurons. Treating the DF cell population with doxorubicin(DOX) followed by incubation in an adipogenesis medium indicated thatstem cells were isolated and differentiated into adipocytes. We haveshown that doxyrubicin (DOX) is effective in enriching the percent stemcells in a cell population, and believe that other cell toxins will alsowork, for example, mitoxantrone (MXR) and acetaldehyde (ACH). We believethat any substrate for the ATP-binding cassette transporters ABCB1 (alsoknown as P-glycoprotein (P-gp) and multi-drug resistance protein (MDR1))and ABCG2 (also known as breast cancer resistance protein (BCRP1) oraldehyde dehydrogenase will work to select the stem cells. Some of thesesubstances are listed in Table 1.

We have also shown that exposure to stresses of heat, salt and lowoxygen will also selectively kill the non-stem cells. We have shown thatexposure of an adult heterogeneous cell population (dental folliclecells) to heat at about 44° C., high salt at about 619 to about 623mmol/kg osmolality, and low oxygen (levels below 21%) resulted in ahigher percentage of stem cells surviving the exposure than the non-stemcells.

Without wishing to be bound by this theory, we believe that the aboveenrichment of stem cells upon exposure to cell toxins or high stressesis due to the self-protective mechanisms that stem cells which enablethem to exist under conditions that normal cells cannot. The number ofstem cells in a heterogeneous population can be enriched by takingadvantage of these self-protective mechanisms and subjecting theheterogeneous population to one or more stress conditions andeliminating the non-stem cells in the population.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A illustrates the results of RT-PCR showing the expression of someABC transporters (BCRP (“Abcg2”) and MDR1 (“Abcb1”)) in vivo in dentalfollicle cells from rats of various ages.

FIG. 1A illustrates the results of RT-PCR showing the expression of someABC transporters (BCRP (“Abcg2”) and MDR1 (“Abcb1”)) in vitro incultured dental follicle stem cells (DFSCs).

FIG. 2 illustrates the percent cell survival rate in rat dental folliclecells as a function of time exposed to 1 uM doxorubicin (DOX).

FIG. 3A depicts rat dental follicle cells (DFCs) incubated in a mediumto stimulate osteogenesis and then stained with Alizarin red stain formineralization to indicate any presence of osteogenesis (or boneformation).

FIG. 3B depicts rat dental follicle stem cells (DFSCs) incubated in amedium to stimulate osteogenesis and then stained with Alizarin redstain for mineralization to indicate any presence of osteogenesis (orbone formation).

FIG. 3C depicts rat dental follicle stem cells (DFSCs) incubated in amedium to stimulate adipogenesis and then stained with Oil Red O stainto indicate the presence of fat tissues. The arrows indicate the stainedadipocytes.

FIG. 3D depicts rat dental follicle stem cells (DFSCs) after incubationin a medium to induce the growth of motor neurons. The arrows indicatethe growth of multipolar neurons.

FIG. 4 illustrates the percent cell survival rate in a heterogeneouspopulation of rat dental follicle cells (DFCs) and dental follicle stemcells (DFSCs) as a function of temperature after exposure for 30minutes.

FIG. 5 compares the proliferation rate of two populations of cellscultured under normal conditions as followed using the Alamar blue cellproliferation assay. The control was a heterogeneous populations ofcells rat dental follicle cells (DFCs) and dental follicle stem cells(DFSCs) that was only cultured at the normal temperature of 37° C. andnever exposed to elevated temperatures. The other population was cellsthat survived the exposure to 44° C. for 30 minutes (primarily DFSCs).

FIG. 6 illustrates the relative increase in expression of selected stemcell-related genes using RT-PCR in putative rat dental follicle stemcells surviving heat shock at 44° C. as compared to the expression ofthe heterogeneous populations of rat dental follicle cells and ratdental follicle stem cells cultured at 37° C.

The current protocols for purification of stem cells from dental tissuesare labor intensive, somewhat vague and often expensive. In contrast, wehave developed safe, simple and efficient techniques to purify stemcells from dental tissues. We believe that one function of stem cells inadult tissues is to serve as repair and regeneration systems to replacedamaged or defective tissues and organs. To fulfill the repair task,stem cells must first survive the original condition causing the damage.Thus we believe that stem cells possess specific or extra defenseprotection mechanisms such as anti-stress or anti-drug mechanisms thatallow them to survive various stresses.

We have treated dental follicle cells with different cell stresses(e.g., cell toxins, heat or osmotic or oxygen stress), and found thatthe surviving cells had a higher potential for differentiation intospecific tissues, for example, adipocytes and bone cells. We have shownthat certain conditions selectively kill non-stem cells and thusincrease the stem cell percentage in a heterogeneous cell population.This method of increasing the percent stem cells in a cell populationcan thus be used to purify stem cells from heterogeneous cellpopulations. To successfully purify stem cells from a heterogeneouspopulation using this method, the stem cells must have unique featuresthat allow them to be distinguished from the non-stem cells. In thisregard, without wishing to be bound by this theory, we believe that thestem cells from dental follicle or pulp are equipped with uniqueanti-stress (including anti-drug) mechanisms that help them to surviveencountered stresses. One or more of the following mechanisms wouldresult in favoring stem cell survival from a stress over non-stem cellssurvival: (1) the mechanism is stem cell specific; (2) the anti-stressmechanism is more active in stem cells than in other cells, e.g., stemcells express significantly higher levels of detoxification enzymes; (3)only stem cells are able to activate or acquire mechanisms to respond tostress; (4) stem cells can boost/fortify their anti-stress mechanism(s)to a higher level than do non-stem cells to respond to given stresses;and (5) the anti-stress mechanisms of stem cells are longer-lasting thanthose of non-stem cells.

Stem cells have been shown to be equipped with a high level of certainABC transporters and ALDHs to detoxify the defined substrate chemicals.We have determined the dosage and duration of doxorubicin (DOX), asubstrate of BCRP1 and MDR1 (both members of ABC transporters), neededto kill the non-stem cells in the cultured dental follicle and dentalpulp cells. We also believe that Mitoxantrone (MXT), another substrateof ABC transporters, could be used to increase the percentage of stemcells. Other substrates of ABC transporters as listed in Table 1 areknown and should work in this method. Similar experiments were conductedwith a substrate of ALDHs, 4-hydroxyperoxycyclopho-sphamide (4-HC), andagain the stem cells were increased in the population. Another substrateof ALDHs that should work in increasing the stem cell percentage isacetaldehyde (ACH). After a given treatment of drug or substrate, thecell survival rates were assessed, and the surviving cells weresubjected to proliferation and differentiation analyses. In all tests,the surviving stem cells were able to proliferate and differentiate.

Alteration of body temperature and shortage of oxygen supply (hypoxia)are frequent medical conditions in humans and other mammals. Severethermal and/or hypoxic stresses can result in cell or tissue damage ordeath. We also have shown that some dental follicle and pulp cells cansurvive thermal or hypoxic stresses, and that the surviving cells havestem cell properties of proliferation and differentiation. In addition,using RT-PCR technology, we have shown that the surviving cells fromheat stress have increased gene expression of stem cell markers over theexpression in control cells not exposed to heat stress. Using PCRarrays, we will compare the expression of the markers between (1) theoriginal cell population and its derivative putative stem cellpopulations (i.e., sub-populations) obtained by different optimizedprotocols; (2) the stem cell sub-population to each other (e.g., are thestem cells derived from BCRP substrate treatment similar to thosederived from hypoxia?). We will combine the gene expression data withproliferation and differentiation data to conduct correlation analysisusing Statistical Analysis Software (SAS). This will characterize thesub-populations surviving from different stresses on a molecular basis,and ultimately will help select the stem cells best suited for tissueengineering.

EXAMPLE 1

Stem Cell Enrichment from Dental Follicle of a Rat

Establishment of cell cultures from rat dental follicle: Dentalfollicles (DF) were surgically removed from the first mandibular molarsof Sprague Dawley rat pups at days 5-7 postnatally, and the isolated DFcells then suspended in MEM medium containing 10% newborn calf serum(NCS), 1 mM sodium pyruvate and antibiotics as previously described(Wise et al., 1992). Next, 5 ml of the cell suspension was transferredinto a T-25 cell culture flask and incubated overnight to allow thecells to adhere. The non-adherent cells then were removed by aspirationand the remaining adherent cells were either grown as previouslydescribed by Wise et al., (1992), which are referred to as “DF cells” or“DFCs” or grown in a stem cell growth medium consisting of α-MEM and 20%heat-inactivated fetal bovine serum (FBS), a culture medium thatencourages the growth of dental follicle stem cells referred to as “DFstem cells” or “DFSCs.” The resulting population of culturing in theα-MEM plus 20% FBS is a heterogeneous cell population containing bothdental follicle cells and dental follicle stem cells (DFSCs), but isreferred to as “DFSCs”. For either culture, upon confluency, the cellswere trypsinized and passaged into new flasks until the desired passageswere obtained.

Expression of ABC transport genes: Total RNA was extracted from the DFCsof the postnatal rat first mandibular molars (day 1 to day 11) and fromthe DFSCs of passage 3 with a TRI REAGENT protocol (Molecular ResearchCenter, Cincinnati, Ohio). The RNA samples were digested with DNase I toremove any possible DNA. RNA concentration was measured by opticaldensity at 260 nm and 280 nm with the OD260/OD280 ratio greater than1.9.

Gene expression of side population stem cell markers, Breast CancerResistant Protein (BCRP; noted as “Abcg2” on FIG. 1) and MultidrugResistant Protein 1 (MDR1, noted as “Abcb1” on FIG. 1), were determinedby conventional RT-PCR. For this, the RNA was reverse-transcribed intocDNA with reverse transcriptase (Invitrogen, Carlsbad, Calif.). The PCRwas conducted by mixing cDNA with gene specific primers, dNTP, PCRbuffer and Taq DNA polymerase followed by running 30 thermal cycles atdenaturing 94° C. 45 sec, annealing 58° C. 1 min and extension 72° C. 1min. PCR was also conducted in parallel to amplify the B-actin gene thatserved as the control. The primer sequences for BCRP (Abcg2) were asfollows: 5′AGTCCGGAAAACAGCTGAGA3′ (forward; SEQ ID NO.1),5′CCCATCACAACGTCATCTTG 3′ (reverse; SEQ ID NO.2). The primer sequencesfor MDRI (Abcb1) were as follows: 5′GGA ACT CTC GCT GCT ATC ATC CAC GGAAC 3′ (forward; SEQ ID NO.3), 5′ACC TGG ATG TAG GCA ACG ATG AGC ACA CC3′(reverse; SEQ ID NO.4). PCR products were electrophoresed in a 1%agarose gel and visualized under UV light.

Cell growth and Differentiation Experiments: To characterize the cellgrowth in culture, DFCs and DFSCs at passage 3 and passage 9 were seededin 3 cm diameter tissue culture Petri dishes and cultured withappropriate media (i.e., DF cells in MEM+10% NCS+sodium pyruvate; DFstem cells in α-MEM+20% FBS). To monitor for stem cells, cultures ofeither DF cells or DF stem cells were fixed in neutral-buffered formalinafter culturing for 1, 3, 5, 7, 9, 11, 13 and 15 days, and then stainedfor alkaline phosphtase (ALP) activity by incubating in ALP substratesolution containing 0.01% Naphthol AS-Mx phosphate for 15 min at roomtemperature. To determine if the cells could differentially pump outHoechst, the substrate of BCRP, Hoechst 33342 was added into the culturemedium at a final concentration of 0.2 μg/ml and then examined under ainverted fluorescence microscope for blue nuclear staining after 20 minof incubation. Unless otherwise noted, the commercial source for thechemicals is Sigma-Aldrich (St. Louis, Mo.).

To determine the differentiation capabilities of the putative stemcells, DFSCs were subjected to various differentiation procedures. Forosteogenesis, cells were cultured for 2 weeks in medium consisting ofDMEM-LG, 10% FBS, 50 μg/ml ascorbate-2 phosphate, 10⁻⁸ M dexamethasone,and 10 mM B-glycerophosphate for 3 weeks as previously described,followed by von Kossa and Alizarin Red staining to assess for mineraldeposition (as described in Hung et al., 2002). For adipogenesis, DFSCswere cultured for 2 weeks in medium consisting of DMEM-LG, 10% FBS(Invitrogen Corporation, Carlsbad, Calif., USA), 50 μg/ml ascorbate-2phosphate (Sigma-Aldrich), 10⁻⁷ M dexamethasone, and 50 μg/mlindomethacin, with a medium change every 3 days (Hung et al., 2002). Thecells then were stained with Oil red O to evaluate adipogenesis.

To induce neurogenesis, DFSCs were initially treated in a transitionmedium consisting of DMEM-LG with 10% FBS and 10 ng/ml basic fibroblastgrowth factor for 24 hours. Next, the cells were incubated in theneuronal induction medium consisting of DMEM-LG, 2% DMSO, 200 μMbutylated hydroxyanisole, 25 mM KCL, 2 mM valporic acid, 10 μMforskolin, 1 μM hydroxycortisone, 5 μg/mL insulin, and 2 mM L-glutamine.After 24 hours of incubation, the cells were fixed and immunostained forneurofilament-200. Briefly, cells were fixed in cold methanol for 5 min,and incubated with anti-neurofilament-200 (Chemicon, Temecula, Calif.)overnight at 4° C. Immunostaining controls were incubated with rabbitIgG instead of primary antibody. Next, they were incubated withsecondary antibody labeled with horseradish peroxidase followed by 3washes with PBS. The horseradish peroxidase was detected by incubationwith 3,3′-diaminobenzidine (DAB) substrate.

When the DFCs were grown in a stem cell growth medium, clusters of cellswere formed after two weeks that were not seen when the cells were grownin normal MEM medium. In turn, these clusters stained for alkalinephosphatase (ALP) which, among other things, is a marker for stem cellswhen it is expressed on the cell membrane. No ALP staining was seen inDF cells grown in normal MEM medium. (Data not shown)

Growing the DF cells in stem cell medium to make the heterogeneous DFSCcell population, and then staining with Hoechst 33342 to detect sidepopulation stem cells showed that some 2-4% of the cells weaklyfluoresced. This indicates these cells are likely stem cells becausestem cells efflux the dye out with BCRP, an ABC membrane transporter. Toconfirm that the BCRP gene was expressed by the cells, conventionalRT-PCR was conducted both in vivo on cells directly taken from thedental follicle and in vitro on the denticle follicle stem cells(DFSCs). The DFs at days 1-11 were examined and all expressed BCRP andMDR1, as did the DFSCs in culture. The results are shown in FIG. 1. BothBCRP and MDR1 expresssion was detected in the DFs of the rat firstmandibular molar at postnatal days 1,2, 5, 7, 9 and 11 in vivo.

To determine the differentiation capabilities of the putative stemcells, cells grown in the stem cell medium (DFSCs) were placed indifferent differentiation media. When placed in an osteogenicdifferentiation medium for 3 weeks, osteoblasts formed and developedmineralization nodules (clusters) as seen by Alizarin red staining andvon Kossa staining FIGS. 3A and 3B show the results of staining withAlizarin red stain. No staining was seen in the DFCs even after grown inosteogenesis medium (FIG. 3A) In contrast, DFSCs grown in the osteogenicmedium, and then stained, showed the red stain indicating bonedeposition or mineralization (FIG. 3B). When cells grown in stem cellmedium (DFSCs) were placed in an adipogenic medium for 2 weeks and thenstained with Oil Red O, stained adipocytes were observed whereas nonewere observed in the cells were not placed in the adipogenic medium.(FIG. 3C). Finally, placing DFSCs into a neuronal induction medium for24 hrs resulted in the development of cells that resembled multipolarneurons. (FIG. 3D) No neurons were seen in the DFSC controls in whichthe cells were not cultured in the neuronal induction medium. Using amarker for late neuron differentiation, neurofilament protein 200,resulted in immunostaining of the neurons in the induction medium withno immunostaining seen in the controls not placed in the neuroninduction medium. Immunostaining controls without primary antibody didnot stain. (Data not shown)

EXAMPLE 2

Initial work with Cell Toxin (DOX) and Thermal Stress

In another experiment to determine to enrich the populations of stemcells, DFSCs were treated with doxorubicin (DOX), a substrate of BCRP,at concentrations of 0.5, 1 or 2 μM for 1, 2, 4 and 6 hrs. The cellswere incubated at 37° C. for the designated duration. After thetreatment, the cells were collected by centrifugation at 800×g for 5min, and the cell pellet washed once with growth medium to completelyremove the substrate. Then the cells were resuspended in 20 ml of mediumand transferred back to the same flask. After 24 hours of incubation,the dead cells were removed by changing the medium, as the viable cellsremained attached on the culture surface. The number of viable cells wascounted in 5 to 10 randomly selected spots (1 mm² each) for calculationof the survival rate as the ratio of treatment/control. The survivingcells were placed in an adipogenesis differentiation medium, aspreviously described.

Treatment of the DFSCs with doxorubicin (DOX) resulted in the number ofsurviving cells decreasing as the duration of DOX (1 μM) incubation wasincreased from 2 hours, to 4 hours and to 6 hours as compared to thecontrol without DOX treatment. The results are shown in FIG. 2. Placingcells that survived treatment with DOX for 4 hours in an adipogenesisinduction medium resulted in the majority of the cells forming Oil Red Opositive adipocytes. In contrast, in the control DFSCs not treated withDOX, but placed in adipogenesis induction medium, the majority of thecells remained undifferentiated with only a few forming adipocytes,(Data not shown). These results indicate that DOX killed most of thenon-stem cells in the DFSC population, and that the surviving stem cellswere still capable of differentiation.

EXAMPLE 3 Heat-shock Enriches Stem Cell Numbers in Dental Follicle CellPopulations

To determine if heat stress could be a means of enriching a populationfor stem cells by eliminating non-stem cells from the dental folliclecell (DFC) population, DFCs were obtained from 1^(st) mandibular molarsof rat pups, as described above. The cells were initially cultured inMEM-α containing 20% FBS to induce the growth of stem cells, and forminga heterogeneous population of non-stem cells and stem cells (DFSCs). Todetermine the tolerance of this heterogeneous population to heat-shock,DFSCs were suspended in normal culture medium and incubated attemperatures from 37° C. (control) to 46° C. for 30 min. The number ofsurviving cells was counted as described above. The results are shown inFIG. 4.

Proliferation assay: The DFSCs surviving from heat shock at 44° C. heatstress and the DFSCs not subjected to heat stress were each seeded intoa 12-well plate at a density of 1000 cells/ml in stem cell growth mediumconsisting of α-MEM+20% FBS. The plates were incubated in normal cultureconditions at 37° C. Cell proliferation was monitored with an alamarblue living cell proliferation assay procedures, as previously described(Yao et al., 2006), at days 1, 4, and 8 during incubation. Alamar bluereduction was calculated. The results are shown in FIG. 5, indicatingthe surviving stem cells were capable of proliferating.

Differentiation assays: To evaluate stem cell properties ofheat-tolerant DFSCs, cells surviving from 44° C. were returned to 37°C., and after 3 to 5 days of growth, were induced for osteogenesis oradipogenesis. The surviving cells were tested using 24-well plates withcells seeded as described above in the proliferation assay section. Foradipogenesis, cells were placed in an adipocyte induction medium, whichconsisted of DMEM-LG supplemented with 10% FBS, 50 μg/ml ascorbate-2phosphate, 10⁻⁷ M dexamethasone, and 50 μg/ml indomethacin, and culturedin a 37° C., 5% CO2 incubator for 2 weeks with a medium change every 3days. The cells were then stained with Oil Red O staining to assess foradipogenesis. For osteogenesis, the induction medium consisted ofDMEM-LG, 10% FBS, 50 μg/ml ascorbate-2 phosphate, 10⁻⁸ M dexamethasone,and 10 mM B-glycerophosphate for 2 weeks as previously described,followed by von Kossa and Alizarin Red staining to assess for mineraldeposition (as described in Hung et al., 2002).

The threshold temperature for elimination of the majority of the cellswas between 44° C. to 44.5° C. (1-6% cells survived; FIG. 4). Cellssurviving from 44° C. grew more rapidly upon returning to 37° C. thandid the control cells as shown in FIG. 5. When those heat-tolerant cellswere placed in a given differentiation medium, both osteogenesis andadipogenesis levels were higher than in the controls not subjected toheat-shock. (Data not shown)

In addition, RT-PCR using a stem cell assay from SABiosciences(SuperArrray Bioscience Corporation, Frederick, Md.) was used to assessthe relative increase in expression of selected stem cell-related genesin putative rat dental follicle stem cells surviving heat shock at 44°C. over the gene expression of the control rat heterogeneous cellpopulation not exposed to heat shock. The results are shown in FIG. 6.

Stem cell gene expression. For the microarray study, we will use a ratstem cell oligo array (SuperArray Bioscience Corporation, Frederick,Md.) containing 256 genes related to the identification, proliferationand differentiation of various stem cells as previously described (Liuand Wise 2007). The array will be processed according to themanufacturer's protocol. Briefly, RNA will be isolated from the putativestem cells after heat exposure and from their counterpart unselectedcells. RNA will then be labeled with biotin-16-dUTP for hybridizationwith the array. Followed by washes to remove unbound cRNA, the cRNAbound to the matching gene specific oligos printed on the membrane willremain. Finally, the bound cRNA on array will be detected usingchemiluminescence, and an image captured with an imaging system. Theimage will be loaded into the GEArray Expression Analysis Suite program(SuperArray Bioscience Corporation) to acquire the intensity of eachgene and then normalized to one of the house-keeping genes (control).The changes in expression of the markers in a given putative stem cellpopulation will be calculated by comparing the given population to itscounterpart unselected population.

Heat-shock was shown to kill mostly non-stem cells, such that theremaining population consisted primarily of stem cells, cells shown tohave increased gene expression for typical stem cell genes. Theremaining stem cells were shown to be capable of differentiation andproliferation.

EXAMPLE 4 Osmotic Stress Enriches Stem Cell Numbers in Dental FollicleCell Populations

A wide range of diseases and medical conditions can cause excessive lossof body fluid and lead to dehydration. Cell dehydration occurs whencells lose water by osmosis. Dehydration can result in tissue damage aswell as cell death. Serving as a repair mechanism, stem cells shouldpossess self protection from dehydration. To determine if stem cellssurvive better in dehydrating condition, a heterogeneous dental folliclecell population (DFSCs) collected and cultured in stem cell medium asdescribed above was treated with a medium of different NaCl osmolalityranging from 404 to 925 mmol/kg. The results indicated that all cellsdied in medium of osmolality greater than 642 mmol/kg. (Data not shown)A small portion of cells survived in medium of osmolality from about 619to about 623 mmol/kg, and these surviving cells showed a greater levelof osteogenesis than the control DFCs when placed into osteogenesisinduction medium for one week. (Data not shown) In addition, cellssurviving from high osmolality medium were smaller in their averagesize. These results indicate that the high osmolality (osmotic stress)enriched the number of stem cells in the remaining population.

EXAMPLE 5 Hypoxia Enriches Stem Cell Numbers in Dental Follicle CellPopulations

To test the effects of hypoxia (low oxygen conditions) on the dentalfollicle cell population, DFSCs obtained as described above were seededin t-25 flasks and cultured in hypoxia chambers containing 1% or 5% O₂for 1 or 2 weeks. The cells cultured at the normal atmospheric condition(about 21% O₂) served as the control. Cell growth was monitored with analamar blue assay as above. After the designated time of incubation, thesurviving cells were subjected to differentiation (osteogenesis andadiopogenesis) and gene expression studies. The results indicated thatDFSCs showed a higher proliferation rate when cultured in 5% oxygen (O₂)as compared to being cultured in normal atmospheric conditions(approximately 21% O₂). In contrast, normal dental follicle cells(non-stem cells) show a similar growth rate in either hypoxia or normalconditions. Moreover, an increase in stem cell characteristics was seenin the dental follicle stem cells grown in 5% O₂. Specifically, dentalfollicle stem cells grown in 5% O₂ exhibited a higher capability ofdifferentiation (e.g., possessing greater adipogenesis potential) thanthe cells grown in normal atmospheric conditions (controls). Alkalinephosphatase (ALP) staining indicated that the dental follicle stem cellssubjected to hypoxic treatment (5% O₂) increased in number, as seen byan increase in the number of ALP-stained cells in comparison to thecontrols (21% O₂). (Data not shown) Moreover, there was an increase inthe amount of ALP-staining of the hypoxia-treated cells, as compared tothe controls. (Data not shown) Increased expression of surface ALP is amarker often used to identify stem cells. Normal dental follicle cellsshow no or very weak ALP staining Thus growing the DFSCs at 5% O₂resulted in an increase in the number of stem cells in the population.

EXAMPLE 6 An ALDH Substrate Enriches Stem Cell Numbers in DentalFollicle Cell Populations

We also tested an ALDH substrate, 4-hydroperoxycyclophosphamide (4-HC),for elimination of non-stem cells in culture. DFSCs were cultured asdescribed above and then treated with 4-HC at the concentrations of 5,10, 15, 20 and 25 μM in α-MEM medium containing 20% FBS for 48 hours at37° C. The majority of the cells were killed at a 4-HC concentrationgreater than 15 μM. The surviving cells then were collected andsubjected to cell proliferation, differentiation, and gene expressionassays as described above for evaluation of stem cell properties.Putative stem cells surviving from a 4-HC treatment expressed a highlevel of multiple stem cell markers, including Abcb1, Prom1, Notch 1,and CD34, as determined by RT-PCR and western blotting. On the average,about a 2 to 4-fold increase in expression of these markers was detectedin the 4-HC surviving cells as compared to the original population.(Data not shown) In addition, 4-HC treatment did not adversely affectthe proliferation of the cells. In fact, flow cytometry analysisindicated that the putative stem cells surviving from the 4-HC treatmenthave a higher proliferation rate than the original cell population notsubjected to 4-HC treatment. When the same 4-HC treatment protocol wasapplied to the dental follicle cells (DFCs; non-stem cells), none ofthem survived.

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The complete disclosures of all references cited in this specificationare hereby incorporated by reference. Also incorporated by reference isthe complete disclosure of the following: S. Yao et al.,“Differentiation of Stem Cells in the Dental Follicle,” Journal ofDental Research, vol. 87 (8), pp. 767-771 (2008). In the event of anotherwise irreconcilable conflict, however, the present specificationshall control.

We claim:
 1. A method to obtain a multipotent stem cell population thatproliferates and differentiates into at least two cell types byincreasing the percentage of stem cells in an adult mammalianheterogeneous cell population, said method comprising the following: A.isolating an adult heterogeneous cell population from a mammal, whereinsaid heterogeneous cell population consists of both multipotent stemcells and non-stem cells, B. exposing the isolated heterogeneous cellpopulation to a stress of high temperature between 44° C. to 44.5° C.,wherein such exposure occurs for duration of time such that the non-stemcells are selectively killed and the surviving cell population has ahigher percentage of stem cells; C. culturing the surviving cellpopulation to increase the number of multipotent stem cells; and D.verifying that the cultured multipotent stem population differentiatesinto two or more cell types selected from the group consisting ofosteoblasts, adipocytes, neurons, and odontoblasts.
 2. The method ofclaim 1, additionally comprising isolating the stem cells from thecultured surviving cell population with the higher percentage of stemcells.
 3. The method of claim 1, wherein the heterogeneous population ofcells is isolated from the group consisting of adult dental follicle,adult dental pulp, blood, skin, gut, testis, and other adult organs. 4.The method of claim 1, wherein the verification step (D) comprisesfurther culturing the proliferated stem cell population from step (C) ina medium that induces cellular differentiation selected from the groupconsisting of an adipogenic medium, an osteogenic medium, a neurogenicmedium, and an odontogenic medium.